Protein Function. chapter

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1 chapter Protein Function 7 Knowing the three-dimensional structure of a protein is an important part of understanding how the protein functions. However, the structure shown in two dimensions on a page is deceptively static. Proteins are dynamic molecules whose functions almost invariably depend on interactions with other molecules, and these interactions are affected in physiologically important ways by sometimes subtle, sometimes striking changes in protein conformation. In this chapter, we explore how proteins interact with other molecules and how their interactions are related to dynamic protein structure. The importance of molecular interactions to a protein s function can hardly be overemphasized. In Chapter 6, we saw that the function of fibrous proteins as structural elements of cells and tissues depends on stable, long-term quaternary interactions between identical polypeptide chains. As we will see in this chapter, the functions of many other proteins involve interactions with a variety of different molecules. Most of these interactions are fleeting, though they may be the basis of complex physiological processes such as oxygen transport, immune function, and muscle contraction, the topics we examine in detail in this chapter. The proteins that carry out these processes illustrate the following key principles of protein function, some of which will be familiar from the previous chapter: The functions of many proteins involve the reversible binding of other molecules. A molecule bound reversibly by a protein is called a ligand. A ligand may be any kind of molecule, including another protein. The transient nature of protein-ligand interactions is critical to life, allowing an organism to respond rapidly and reversibly to changing environmental and metabolic circumstances. A ligand binds at a site on the protein called the binding site, which is complementary to the ligand in size, shape, charge, and hydrophobic or hydrophilic character. Furthermore, the interaction is specific: the protein can discriminate among the thousands of different molecules in its environment and selectively bind only one or a few. A given protein may have separate binding sites for several different ligands. These specific molecular interactions are crucial in maintaining the high degree of order in a living system. (This discussion excludes the binding of water, which may interact weakly and nonspecifically with many parts of a protein. In Chapter 8, we consider water as a specific ligand for many enzymes.) Proteins are flexible. Changes in conformation may be subtle, reflecting molecular vibrations and small movements of amino acid residues 203

2 204 Part II Structure and Catalysis throughout the protein. A protein flexing in this way is sometimes said to breathe. Changes in conformation may also be quite dramatic, with major segments of the protein structure moving as much as several nanometers. Specific conformational changes are frequently essential to a protein s function. The binding of a protein and ligand is often coupled to a conformational change in the protein that makes the binding site more complementary to the ligand, permitting tighter binding. The structural adaptation that occurs between protein and ligand is called induced fit. In a multisubunit protein, a conformational change in one subunit often affects the conformation of other subunits. Interactions between ligands and proteins may be regulated, usually through specific interactions with one or more additional ligands. These other ligands may cause conformational changes in the protein that affect the binding of the first ligand. Enzymes represent a special case of protein function. Enzymes bind and chemically transform other molecules they catalyze reactions. The molecules acted upon by enzymes are called reaction substrates rather than ligands, and the ligand-binding site is called the catalytic site or active site. In this chapter we emphasize the noncatalytic functions of proteins. In Chapter 8 we consider catalysis by enzymes, a central topic in biochemistry. You will see that the themes of this chapter binding, specificity, and conformational change are continued in the next chapter, with the added element of proteins acting as reactants in chemical transformations. Reversible Binding of a Protein to a igand: Oxygen-Binding Proteins Myoglobin and hemoglobin may be the most-studied and best-understood proteins. They were the first proteins for which three-dimensional structures were determined, and our current understanding of myoglobin and hemoglobin is garnered from the work of thousands of biochemists over several decades. Most important, they illustrate almost every aspect of that most central of biochemical processes: the reversible binding of a ligand to a protein. This classic model of protein function will tell us a great deal about how proteins work. Oxygen Can Be Bound to a Heme Prosthetic Group Oxygen is poorly soluble in aqueous solutions (see Table 4 3) and cannot be carried to tissues in sufficient quantity if it is simply dissolved in blood serum. Diffusion of oxygen through tissues is also ineffective over distances greater than a few millimeters. The evolution of larger, multicellular animals depended on the evolution of proteins that could transport and store oxygen. However, none of the amino acid side chains in proteins is suited for the reversible binding of oxygen molecules. This role is filled by certain transition metals, among them iron and copper, that have a strong tendency to bind oxygen. Multicellular organisms exploit the properties of metals, most commonly iron, for oxygen transport. However, free iron promotes the formation of highly reactive oxygen species such as hydroxyl radicals that can damage DNA and other macromolecules. Iron used in cells is therefore bound in forms that sequester it and/or make it less reactive. In multicellular organisms especially those in which iron, in its oxygen-carrying capacity, must be transported over large distances iron is often incorporated into a proteinbound prosthetic group called heme. (A prosthetic group is a compound permanently associated with a protein that contributes to the protein s function.)

3 Chapter 7 Protein Function 205 figure 7 1 Heme. The heme group is present in myoglobin, hemoglobin, and many other proteins, designated heme proteins. Heme consists of a complex organic ring structure, protoporphyrin IX, to which is bound an iron atom in its ferrous (Fe 2 ) state. Porphyrins, of which protoporphyrin IX is only one example, consist of four pyrrole rings linked by methene bridges (a), with substitutions at one or more of the positions denoted X. Two representations of heme are shown in (b) and (c). The iron atom of heme has six coordination bonds: four in the plane of, and bonded to, the flat porphyrin ring system, and two perpendicular to it (d). X X X X N NH HN N X X (a) X X Heme (or haem) consists of a complex organic ring structure, protoporphyrin, to which is bound a single iron atom in its ferrous (Fe 2 ) state (Fig. 7 1). The iron atom has six coordination bonds, four to nitrogen atoms that are part of the flat porphyrin ring system and two perpendicular to the porphyrin. The coordinated nitrogen atoms (which have an electron-donating character) help prevent conversion of the heme iron to the ferric (Fe 3 ) state. Iron in the Fe 2 state binds oxygen reversibly; in the Fe 3 state it does not bind oxygen. Heme is found in a number of oxygentransporting proteins, as well as in some proteins, such as the cytochromes, that participate in oxidation-reduction (electron transfer) reactions (Chapter 19). In free heme molecules, reaction of oxygen at one of the two open coordination bonds of iron (perpendicular to the plane of the porphyrin molecule, above and below) can result in irreversible conversion of Fe 2 to Fe 3. In heme-containing proteins, this reaction is prevented by sequestering the heme deep within a protein structure where access to the two open coordination bonds is restricted. One of these two coordination bonds is occupied by a side-chain nitrogen of a His residue. The other is the binding site for molecular oxygen (O 2 ) (Fig. 7 2). When oxygen binds, the electronic properties of heme iron change; this accounts for the change in color from the dark purple of oxygen-depleted venous blood to the bright red of oxygen-rich arterial blood. Some small molecules, such as carbon monoxide (CO) and nitric oxide (NO), coordinate to heme iron with greater affinity than does O 2. When a molecule of CO is bound to heme, O 2 is excluded, which is why CO is highly toxic to aerobic organisms. By surrounding and sequestering heme, oxygen-binding proteins regulate the access of CO and other small molecules to heme iron. O CH 3 CH O CH 2 O O C C CH 2 CH 2 CH 2 C CH C CH 3 C C C C CH 3 C N N C CH Fe CH C N N C CH C C C C CH 3 CH 2 C CH C (c) (b) CH 2 Fe (d) Edge view N CH N Fe O 2 C CH 2 CH Histidine residue Plane of porphyrin ring system figure 7 2 The heme group viewed from the side. This view shows the two coordination bonds to Fe 2 perpendicular to the porphyrin ring system. One of these two bonds is occupied by a His residue, sometimes called the proximal His. The other is the binding site for oxygen. The remaining four coordination bonds are in the plane of, and bonded to, the flat porphyrin ring system.

4 206 Part II Structure and Catalysis Myoglobin Has a Single Binding Site for Oxygen Myoglobin (M r 16,700; abbreviated Mb) is a relatively simple oxygenbinding protein found in almost all mammals, primarily in muscle tissue. It is particularly abundant in the muscles of diving mammals such as seals and whales that must store enough oxygen for prolonged excursions undersea. Proteins very similar to myoglobin are widely distributed, occurring even in some single-celled organisms. Myoglobin stores oxygen for periods when energy demands are high and facilitates its distribution to oxygen-starved tissues. Myoglobin is a single polypeptide of 153 amino acid residues with one molecule of heme. It is typical of the family of proteins called globins, which have similar primary and tertiary structures. The polypeptide is made up of eight a-helical segments connected by bends (Fig. 7 3). About 78% of the amino acid residues in the protein are found in these a helices. Any detailed discussion of protein function inevitably involves protein structure. Our treatment of myoglobin will be facilitated by introducing some structural conventions peculiar to globins. As seen in Figure 7 3, the helical segments are labeled A through H. An individual amino acid residue may be designated either by its position in the amino acid sequence or by its location within the sequence of a particular a-helical segment. For example, the His residue coordinated to the heme in myoglobin, His 93 (the 93rd amino acid residue from the amino-terminal end of the myoglobin polypeptide sequence), is also called His F8 (the 8th residue in a helix F). The bends in the structure are labeled AB, CD, EF, and so forth, reflecting the a-helical segments they connect. Protein-igand Interactions Can Be Described Quantitatively The function of myoglobin depends on the protein s ability not only to bind oxygen, but also to release it when and where it is needed. Function in biochemistry often revolves around a reversible protein-ligand interaction of this type. A quantitative description of this interaction is therefore a central part of many biochemical investigations. C CD FG B D F figure 7 3 The structure of myoglobin. The eight a-helical segments (shown here as cylinders) are labeled A through H. Nonhelical residues in the bends that connect them are labeled AB, CD, EF, and so forth, indicating the segments they interconnect. A few bends, including BC and DE, are abrupt and do not contain any residues; these are not normally labeled. (The short segment visible between D and E is an artifact of the computer representation.) The heme is bound in a pocket made up largely of the E and F helices, although amino acid residues from other segments of the protein also participate. H EF A G E GH AB

5 Chapter 7 Protein Function 207 In general, the reversible binding of a protein (P) to a ligand () can be described by a simple equilibrium expression: P 34 P The reaction is characterized by an equilibrium constant, K a, such that (7 1) K a [P] [P][] (7 2) The term K a is an association constant (not to be confused with the K a that denotes an acid dissociation constant; see p. 98). The association constant provides a measure of the affinity of the ligand for the protein. K a has units of M 1 ; a higher value of K a corresponds to a higher affinity of the ligand for the protein. A rearrangement of Equation 7 2 shows that the ratio of bound to free protein is directly proportional to the concentration of free ligand: K a [] [P] [P] (7 3) When the concentration of the ligand is much greater than the concentration of ligand-binding sites, the binding of the ligand by the protein does not appreciably change the concentration of free (unbound) ligand that is, [] remains constant. This condition is broadly applicable to most ligands that bind to proteins in cells and simplifies our description of the binding equilibrium. Thus we can consider the binding equilibrium from the standpoint of the fraction, v (theta), of ligand-binding sites on the protein that are occupied by ligand: v binding sites occupied total binding sites (7 4) Substituting K a [][P] for [P] (see Eqn 7 3) and rearranging terms gives K v a [][P] K a [][P] [P] K a [] K a [] 1 [] [] 1 K a (7 5) The term K a can be determined from a plot of v versus the concentration of free ligand, [] (Fig. 7 4a). Any equation of the form x y/(y z) describes a hyperbola, and v is thus found to be a hyperbolic function of []. The fraction of ligand-binding sites occupied approaches saturation asymptotically as [] increases. The [] at which half of the available ligand-binding sites are occupied (at v 0.5) corresponds to 1/K a. [P] [P] [P] figure 7 4 Graphical representations of ligand binding. The fraction of ligand-binding sites occupied, v, is plotted against the concentration of free ligand. Both curves are rectangular hyperbolas. (a) A hypothetical binding curve for a ligand. The [] at which half of the available ligandbinding sites are occupied is equivalent to 1/K a, or K d. The curve has a horizontal asymptote at v 1 and a vertical asymptote (not shown) at [] 1/K a. (b) A curve describing the binding of oxygen to myoglobin. The partial pressure of O 2 in the air above the solution is expressed in terms of kilopascals (kpa). Oxygen binds tightly to myoglobin with a P 50 of only 0.26 kpa v 0.5 v K d 5 10 [] (arbitrary units) (a) 0 P po 2 (kpa) (b)

6 208 Part II Structure and Catalysis It is sometimes intuitively simpler to consider the dissociation constant, K d, which is the reciprocal of K a (K d 1/K a ) and is given in units of molar concentration (M). K d is the equilibrium constant for the release of ligand. The relevant expressions change to K d [P][] [P] [P] [P][] K d v [] [] K d (7 6) (7 7) (7 8) When [] is equal to K d, half of the ligand-binding sites are occupied. When [] is lower than K d, little ligand binds to the protein. In order for 90% of the available ligand-binding sites to be occupied, [] must be nine times greater than K d. In practice, K d is used much more often than K a to express the affinity of a protein for a ligand. Note that a lower value of K d corresponds to a higher affinity of ligand for the protein. The mathematics can be reduced to simple statements: K d is the molar concentration of ligand at which half of the available ligand-binding sites are occupied. At this point, the protein is said to have reached half saturation with respect to ligand binding. The more tightly a protein binds a ligand, the lower the concentration of ligand required for half the binding sites to be occupied, and thus the lower the value of K d. Some representative dissociation constants are given in Table 7 1. The binding of oxygen to myoglobin follows the patterns discussed above, but because oxygen is a gas, we must make some minor adjustments to the equations. We can simply substitute the concentration of dissolved oxygen for [] in Equation 7 8 to give v [O 2 ] [O 2 ] K d (7 9) As for any ligand, K d is equal to the [O 2 ] at which half of the available ligandbinding sites are occupied, or [O 2 ] 0.5. Equation 7 9 becomes [O v 2 ] [O 2 ] [O 2 ] 0.5 (7 10) table 7 1 Some Protein Dissociation Constants Protein igand K d (M)* Avidin (egg white) Biotin Insulin receptor (human) Insulin Anti-HIV immunoglobulin gp41 (HIV-1 surface (human) protein) Nickel-binding protein (E. coli) Ni Calmodulin (rat) Ca *A reported dissociation constant is valid only for the particular solution conditions under which it was measured. K d values for a protein-ligand interaction can be altered, sometimes by several orders of magnitude, by changes in solution salt concentration, ph, or other variables. Interaction of avidin with the enzymatic cofactor biotin is among the strongest noncovalent biochemical interactions known. This immunoglobulin was isolated as part of an effort to develop a vaccine against HIV. Immunoglobulins (described later in the chapter) are highly variable, and the K d reported here should not be considered characteristic of all immunoglobulins. Calmodulin has four binding sites for calcium. The values shown reflect the highest- and lowest-affinity binding sites observed in one set of measurements.

7 Chapter 7 Protein Function 209 The concentration of a volatile substance in solution, however, is always proportional to its partial pressure in the gas phase above the solution. In experiments using oxygen as a ligand, it is the partial pressure of oxygen, po 2, that is varied because this is easier to measure than the concentration of dissolved oxygen. If we define the partial pressure of oxygen at [O 2 ] 0.5 as P 50, substitution in Equation 7 10 gives po v 2 po 2 P 50 (7 11) A binding curve for myoglobin that relates v to po 2 is shown in Figure 7 4b. Protein Structure Affects How igands Bind The binding of a ligand to a protein is rarely as simple as the above equations would suggest. The interaction is greatly affected by protein structure and is often accompanied by conformational changes. For example, the specificity with which heme binds its various ligands is altered when the heme is a component of myoglobin. CO binds to free heme molecules over 20,000 times better than does O 2 (the K d or P 50 for CO binding is more than 20,000 times lower than that for O 2 ) but binds only about 200 times better when the heme is bound in myoglobin. The difference is partly explained by steric hindrance. When O 2 binds to free heme, the axis of the oxygen molecule is positioned at an angle to the FeXO bond (Fig. 7 5a). In contrast, when CO binds to free heme, the Fe, C, and O atoms lie in a straight line (Fig. 7 5b). In both cases, the binding reflects the geometry of hybrid orbitals in each ligand. In myoglobin, His 64 (His E7), on the O 2 -binding side of the heme, is too far away to coordinate with the heme iron, but it does interact with a ligand bound to heme. This residue, called the distal His, does not affect the binding of O 2 (Fig. 7 5c) but may preclude the linear binding of CO, providing one explanation for the diminished binding of CO to heme in myoglobin (and hemoglobin). This effect on CO binding is physiologically important, because CO is a low-level byproduct of cellular metabolism. Other factors, not yet well-defined, also seem to modulate the interaction of heme with CO in these proteins. The binding of O 2 to the heme in myoglobin also depends on molecular motions, or breathing, in the protein structure. The heme molecule is deeply buried in the folded polypeptide, with no direct path for oxygen to go from the surrounding solution to the ligand-binding site. If the protein were rigid, O 2 could not enter or leave the heme pocket at a measurable Val E11 rate. However, rapid molecular flexing of the amino acid side chains produces transient cavities in the protein structure, and O 2 evidently makes its way in and out by moving through these cavities. Computer simulations of rapid structural fluctuations in myoglobin suggest that there are many such pathways. One major route is provided by rotation of the side chain of the distal His (His 64 ), which occurs on a nanosecond (10 9 s) time scale. Even subtle conformational changes can be critical for protein activity. figure 7 5 Steric effects on the binding of ligands to the heme of myoglobin. (a) Oxygen binds to heme with the O 2 axis at an angle, a binding conformation readily accommodated by myoglobin. (b) Carbon monoxide binds to free heme with the CO axis perpendicular to the plane of the porphyrin ring. CO binding to the heme in myoglobin is forced to adopt a slight angle because the perpendicular arrangement is sterically blocked by His E7, the distal His. This effect weakens the binding of CO to myoglobin. (c) Another view showing the arrangement of key amino acid residues around the heme of myoglobin. The bound O 2 is hydrogen-bonded to the distal His, His E7 (His 64 ), further facilitating the binding of O 2. H O 2 Fe His E7 His F8 Phe CD1 O O J A OFeO A X (a) O c C A OFeO A X (b) (c)

8 210 Part II Structure and Catalysis Oxygen Is Transported in Blood by Hemoglobin Nearly all the oxygen carried by whole blood in animals is bound and transported by hemoglobin in erythrocytes (red blood cells). Normal human erythrocytes are small (6 to 9 mm in diameter), biconcave disks. They are formed from precursor stem cells called hemocytoblasts. In the maturation process, the stem cell produces daughter cells that form large amounts of hemoglobin and then lose their intracellular organelles nucleus, mitochondria, and endoplasmic reticulum. Erythrocytes are thus incomplete, vestigial cells, unable to reproduce and, in humans, destined to survive for only about 120 days. Their main function is to carry hemoglobin, which is dissolved in the cytosol at a very high concentration ( 34% by weight). In arterial blood passing from the lungs through the heart to the peripheral tissues, hemoglobin is about 96% saturated with oxygen. In the venous blood returning to the heart, hemoglobin is only about 64% saturated. Thus, each 100 m of blood passing through a tissue releases about onethird of the oxygen it carries, or 6.5 m of O 2 gas at atmospheric pressure and body temperature. Myoglobin, with its hyperbolic binding curve for oxygen (Fig. 7 4b), is relatively insensitive to small changes in the concentration of dissolved oxygen and so functions well as an oxygen-storage protein. Hemoglobin, with its multiple subunits and O 2 -binding sites, is better suited to oxygen transport. As we will see, interactions between the subunits of a multimeric protein can permit a highly sensitive response to small changes in ligand concentration. Interactions among the subunits in hemoglobin cause conformational changes that alter the affinity of the protein for oxygen. The modulation of oxygen binding allows the O 2 -transport protein to respond to changes in oxygen demand by tissues. Hemoglobin Subunits Are Structurally Similar to Myoglobin Hemoglobin (M r 64,500; abbreviated Hb) is roughly spherical, with a diameter of nearly 5.5 nm. It is a tetrameric protein containing four heme prosthetic groups, one associated with each polypeptide chain. Adult hemoglobin contains two types of globin, two a chains (141 residues each) and two b chains (146 residues each). Although fewer than half of the amino acid residues in the polypeptide sequences of the a and b subunits are identical, the three-dimensional structures of the two types of subunits are very similar. Furthermore, their structures are very similar to that of myoglobin (Fig. 7 6), even though the amino acid sequences of the three polypeptides are identical at only 27 positions (Fig. 7 7). All three polypeptides are Heme group figure 7 6 A comparison of the structures of myoglobin and the b subunit of hemoglobin. Myoglobin b subunit of hemoglobin

9 Mb Hb Hb Mb Hb Hb Mb Hb Hb NA1 1 V 1 V 1V E P F F H A D E K R E A F 100 A1 S S T M V I E P P K M S S G G A E D7 A G G E H N E D E E1 S S N A C V W K K E A P I Q T S 60 D Q K I V N A V 60 V H V C V V V K K K V T V K T K G A H A A Distal His E7 H H H H A A V A G G G S A H W W W V 60 K K G19 R H H A G G T K K H F K K K V V V 120 P P G V V V A G A 120 K A16 E G T D G D E E A A A A A F F F B1 20 D 20 H N F H1 G T T V A 20 V G T S A P P A G D A N D D 120 A P G E E I A G A V V H Y V E19 V Q H Q G G G K A A G A A Q A G K H H A S A D E E K V M Y I A A 80 G D D N D Q H D 80 N K K K I E G H M A F V R R R E P K V M A N G E A A F F E A T S G K V F1 80 F F V V B16 S S V K S A R S A C1 H F Y P A T 140 K T N P P P D V 140 A E T W A S S I T T T Q D E A T A K Q S H21 A S H E T 40 R Proximal His F8 H H H K K K HC1 C7 K Y F F9 A A C Y 140 Y Y HC2 F F F T H D K 141 R 146 H HC3 D P E K K K E R H S H H26 F F F K R H G K G I V V Y H D D G1 100 P D D Q I P 100 P 153 G K S S K V E D1 T H T Y N N figure 7 7 The amino acid sequences of whale myoglobin and the a and b chains of human hemoglobin. Dashed lines mark helix boundaries. To align the sequences optimally, short breaks must be incorporated into both Hb sequences where a few amino acids are present in the other sequences. With the exception of the missing D helix in Hba, this alignment permits the use of the helix lettering convention that emphasizes the common positioning of amino acid residues that are identical in all three structures (shaded). Residues shaded in red are conserved in all known globins. Note that a common letter-and-number designation for amino acids in two or three different structures does not necessarily correspond to a common position in the linear sequence of amino acids in the polypeptides. For example, the distal His residue is His E7 in all three structures, but corresponds to His 64, His 58, and His 63 in the linear sequences of Mb, Hba, and Hbb, respectively. Nonhelical residues at the amino and carboxyl termini, beyond the first (A) and last (H) a-helical segments, are labeled NA and HC, respectively. Hb and Hb only 211

10 212 Part II Structure and Catalysis a 2 b 1 members of the globin family of proteins. The helix-naming convention described for myoglobin is also applied to the hemoglobin polypeptides, except that the a subunit lacks the short D helix. The heme-binding pocket is made up largely of the E and F helices. The quaternary structure of hemoglobin features strong interactions between unlike subunits. The a 1 b 1 interface (and its a 2 b 2 counterpart) involves over 30 residues and is sufficiently strong that although mild treatment of hemoglobin with urea tends to cause the tetramer to disassemble into ab dimers, the dimers remain intact. The a 1 b 2 (and a 2 b 1 ) interface involves 19 residues (Fig. 7 8). Hydrophobic interactions predominate at the interfaces, but there are also many hydrogen bonds and a few ion pairs (sometimes referred to as salt bridges), whose importance is discussed below. b 2 figure 7 8 Dominant interactions between hemoglobin subunits. In this representation, a subunits are light and b subunits are dark. The strongest subunit interactions, highlighted, occur between unlike subunits. When oxygen binds, the a 1 b 1 contact changes little, but there is a large change at the a 1 b 2 contact, with several ion pairs broken. a 1 Hemoglobin Undergoes a Structural Change on Binding Oxygen X-ray analysis has revealed two major conformations of hemoglobin: the R state and the T state. Although oxygen binds to hemoglobin in either state, it has a significantly higher affinity for hemoglobin in the R state. Oxygen binding stabilizes the R state. When oxygen is absent experimentally, the T state is more stable and is thus the predominant conformation of deoxyhemoglobin. T and R originally denoted tense and relaxed, respectively, because the T state is stabilized by a greater number of ion pairs, many of which lie at the a 1 b 2 (and a 2 b 1 ) interface (Fig. 7 9). The binding Asp FG1 b subunit a subunit ys C5 His HC3 (a) figure 7 9 Some ion pairs that stabilize the T state of deoxyhemoglobin. (a) A close-up view of a portion of a deoxyhemoglobin molecule in the T state. Interactions between the ion pairs His HC3 and Asp FG1 of the b subunit (blue) and between ys C5 of the a subunit (gray) and the a-carboxyl group of His HC3 of the b subunit are shown with dashed lines. (Recall that HC3 is the carboxylterminal residue of the b subunit.) (b) The interactions between these ion pairs and others not shown in (a) are schematized in this representation of the extended polypeptide chains of hemoglobin. NH 3 COO NH 3 COO Arg Asp HC3 H9 ys C5 His Asp HC3 FG1 b 2 a 1 a 2 b 1 (b) Asp FG1 Asp H9 His HC3 ys C5 Arg HC3 COO NH 3 COO NH 3

11 Chapter 7 Protein Function 213 His HC3 a 2 b 1 a 2 b 1 His HC3 b 2 a 1 b 2 a 1 His HC3 T state of O 2 to a hemoglobin subunit in the T state triggers a change in conformation to the R state. When the entire protein undergoes this transition, the structures of the individual subunits change little, but the ab subunit pairs slide past each other and rotate, narrowing the pocket between the b subunits (Fig. 7 10). In this process, some of the ion pairs that stabilize the T state are broken and some new ones are formed. Max Perutz proposed that the T 88n R transition is triggered by changes in the positions of key amino acid side chains surrounding the heme. In the T state, the porphyrin is slightly puckered, causing the heme iron to protrude somewhat on the proximal His (His F8) side. The binding of O 2 causes the heme to assume a more planar conformation, shifting the position of the proximal His and the attached F helix (Fig. 7 11). Also, a Val residue in the E helix (Val E11) partially blocks the heme in the T state and must swing out of the way for oxygen to bind (Fig. 7 10). These changes lead to adjustments in the ion pairs at the a 1 b 2 interface. R state figure 7 10 The T 88n R transition. In these depictions of deoxyhemoglobin, as in Figure 7 9, the b subunits are light blue and the a subunits are gray. Positively charged side chains and chain termini involved in ion pairs are shown in blue, their negatively charged partners in pink. The ys C5 of each a subunit and Asp FG1 of each b subunit are visible but not labeled (compare Fig. 7 9a). Note that the molecule is oriented slightly differently than in Figure 7 9. The transition from the T state to the R state shifts the subunit pairs substantially, affecting certain ion pairs. Most noticeably, the His HC3 residues at the carboxyl termini of the b subunits, which are involved in ion pairs in the T state, rotate in the R state toward the center of the molecule where they are no longer in ion pairs. Another dramatic result of the T 88n R transition is a narrowing of the pocket between the b subunits. Val FG5 eu FG3 Heme O 2 His F8 T state Helix F eu F4 R state figure 7 11 Changes in conformation near heme on O 2 binding. The shift in the position of the F helix when heme binds O 2 is one of the adjustments that is believed to trigger the T 88n R transition.

12 214 Part II Structure and Catalysis v po 2 in tissues High-affinity state Transition from low- to highaffinity state po 2 in lungs figure 7 12 A sigmoid (cooperative) binding curve. A sigmoid binding curve can be viewed as a hybrid curve reflecting a transition from a low-affinity to a high-affinity state. Cooperative binding, as manifested by a sigmoid binding curve, renders hemoglobin more sensitive to the small differences in O 2 concentration between the tissues and the lungs, allowing hemoglobin to bind oxygen in the lungs where po 2 is high and release it in the tissues where po 2 is low ow-affinity state po 2 (kpa) Hemoglobin Binds Oxygen Cooperatively Hemoglobin must bind oxygen efficiently in the lungs, where the po 2 is about 13.3 kpa, and release oxygen in the tissues, where the po 2 is about 4 kpa. Myoglobin, or any protein that binds oxygen with a hyperbolic binding curve, would be ill-suited to this function, for the reason illustrated in Figure A protein that bound O 2 with high affinity would bind it efficiently in the lungs but would not release much of it in the tissues. If the protein bound oxygen with a sufficiently low affinity to release it in the tissues, it would not pick up much oxygen in the lungs. Hemoglobin solves the problem by undergoing a transition from a lowaffinity state (the T state) to a high-affinity state (the R state) as more O 2 molecules are bound. As a result, hemoglobin has a hybrid S-shaped, or sigmoid, binding curve for oxygen (Fig. 7 12). A single-subunit protein with a single ligand-binding site cannot produce a sigmoid binding curve even if binding elicits a conformational change because each molecule of ligand binds independently and cannot affect the binding of another molecule. In contrast, O 2 binding to individual subunits of hemoglobin can alter the affinity for O 2 in adjacent subunits. The first molecule of O 2 that interacts with deoxyhemoglobin binds weakly, because it binds to a subunit in the T state. Its binding, however, leads to conformational changes that are communicated to adjacent subunits, making it easier for additional molecules of O 2 to bind. In effect, the T 88n R transition occurs more readily in the second subunit once O 2 is bound to the first subunit. The last (fourth) O 2 molecule binds to a heme in a subunit that is already in the R state, and hence it binds with much higher affinity than the first molecule. An allosteric protein is one in which the binding of a ligand to one site affects the binding properties of another site on the same protein. The term allosteric derives from the Greek allos, other, and stereos, solid or shape. Allosteric proteins are those having other shapes or conformations induced by the binding of ligands referred to as modulators. The conformational changes induced by the modulator(s) interconvert more-active and less-active forms of the protein. The modulators for allosteric proteins may be either inhibitors or activators. When the normal ligand and modulator are identical, the interaction is termed homotropic. When the modulator is a molecule other than the normal ligand the interaction is heterotropic. Some proteins have two or more modulators and therefore can have both homotropic and heterotropic interactions. Cooperative binding of a ligand to a multimeric protein, such as we observe with the binding of O 2 to hemoglobin, is a form of allosteric binding often observed in multimeric proteins. The binding of one ligand affects the affinities of any remaining unfilled binding sites, and O 2 can be considered as both a normal ligand and an activating homotropic modulator. There is only one binding site for O 2 on each subunit, so the allosteric effects giving rise to cooperativity are mediated by conformational changes transmitted from one subunit to another by subunit-subunit interactions. A sigmoid

13 Chapter 7 Protein Function 215 bonding curve is diagnostic of cooperative binding. It permits a much more sensitive response to ligand concentration and is important to the function of many multisubunit proteins. The principle of allostery extends readily to regulatory enzymes, as we will see in Chapter 8. Cooperative igand Binding Can Be Described Quantitatively Cooperative binding of oxygen by hemoglobin was first analyzed by Archibald Hill in For a protein with n binding sites, the equilibrium of Equation 7 1 becomes P n 34 P n and the expression for the association constant becomes K a [P n ] [P][] n (7 12) (7 13) The expression for v (see Eqn 7 8) is v []n [] n K d Rearranging, then taking the log of both sides, yields (7 14) v []n 1 v K d log v 1 v n log [] log K d (7 15) (7 16) Equation 7 16 is the Hill equation, and a plot of log [v/(1 v)] versus log [] is called a Hill plot. Based on the equation, the Hill plot should have a slope of n. However, the experimentally determined slope actually reflects not the number of binding sites, but the degree of interaction between them. The slope of a Hill plot is therefore denoted n H, the Hill coefficient, which is a measure of the degree of cooperativity. If n H equals 1, ligand binding is not cooperative, a situation that can arise even in a multisubunit protein if the subunits do not communicate. An n H of greater than 1 indicates positive cooperativity in ligand binding. This is the situation observed in hemoglobin, in which the binding of one molecule of ligand facilitates the binding of others. The theoretical upper limit for n H is reached when n H n. In this case the binding would be completely cooperative: all binding sites on the protein would bind ligand simultaneously, and no protein molecules partially saturated with ligand would be present under any conditions. This limit is never reached in practice, and the measured value of n H is always less than the actual number of ligand-binding sites in the protein. An n H of less than 1 indicates negative cooperativity, in which the binding of one molecule of ligand impedes the binding of others. Welldocumented cases of negative cooperativity are rare. To adapt the Hill equation to the binding of oxygen to hemoglobin we must again substitute po 2 for [] and P 50 for K d : log v 1 v n log po 2 log P 50 Hill plots for myoglobin and hemoglobin are given in Figure (7 17) ) 1 ( log Myoglobin n H 1 Hemoglobin high-affinity state n H 1 Hemoglobin n H 3 Hemoglobin low-affinity state n H log po 2 figure 7 13 Hill plots for the binding of oxygen to myoglobin and hemoglobin. When n H 1, there is no evident cooperativity. The maximum degree of cooperativity observed for hemoglobin corresponds approximately to n H 3. Note that while this indicates a high level of cooperativity, n H is less than n, the number of O 2 -binding sites in hemoglobin. This is normal for a protein that exhibits allosteric binding behavior. Two Models Suggest Mechanisms for Cooperative Binding Biochemists now know a great deal about the T and R states of hemoglobin, but much remains to be learned about how the T 88n R transition occurs. Two models for the cooperative binding of ligands to proteins with multiple binding sites have greatly influenced thinking about this problem.

14 216 Part II Structure and Catalysis figure 7 14 Two general models for the interconversion of inactive and active forms of cooperative ligand-binding proteins. Although the models may be applied to any protein including any enzyme (Chapter 8) that exhibits cooperative binding, four subunits are shown because the model was originally proposed for hemoglobin. In the concerted, or all-or-none, model (a) all the subunits are postulated to be in the same conformation, either all (low affinity or inactive) or all (high affinity or active). Depending on the equilibrium, K 1, between and forms, the binding of one or more ligand molecules () will pull the equilibrium toward the form. Subunits with bound are shaded. In the sequential model (b) each individual subunit can be in either the or form. A very large number of conformations is thus possible. All All (a) (b) The first model was proposed by Jacques Monod, Jeffries Wyman, and Jean-Pierre Changeux in 1965, and is called the MWC model or the concerted model (Fig. 7 14a). The concerted model assumes that the subunits of a cooperatively binding protein are functionally identical, that each subunit can exist in (at least) two conformations, and that all subunits undergo the transition from one conformation to the other simultaneously. In this model, no protein has individual subunits in different conformations. The two conformations are in equilibrium. The ligand can bind to either conformation, but binds each with different affinity. Successive binding of ligand molecules to the low-affinity conformation (which is more stable in the absence of ligand) makes a transition to the high-affinity conformation more likely. In the second model, the sequential model (Fig. 7 14b), proposed in 1966 by Daniel Koshland and colleagues, ligand binding can induce a change of conformation in an individual subunit. A conformational change in one subunit makes a similar change in an adjacent subunit, as well as the binding of a second ligand molecule, more likely. There are more potential intermediate states in this model than in the concerted model. The two models are not mutually exclusive; the concerted model may be viewed as the all-or-none limiting case of the sequential model. In Chapter 8 we will use these models when we investigate allosteric enzymes. Hemoglobin Also Transports H and CO 2 In addition to carrying nearly all the oxygen required by cells from the lungs to the tissues, hemoglobin carries two end products of cellular respiration H and CO 2 from the tissues to the lungs and the kidneys, where they are excreted. The CO 2, produced by oxidation of organic fuels in mitochondria, is hydrated to form bicarbonate: CO 2 H 2 O 34 H HCO 3 This reaction is catalyzed by carbonic anhydrase, an enzyme particularly abundant in erythrocytes. Carbon dioxide is not very soluble in aqueous solution, and bubbles of CO 2 would form in the tissues and blood if it were not converted to bicarbonate. As you can see from the equation, the hydration of CO 2 results in an increase in the H concentration (a decrease in ph) in

15 Chapter 7 Protein Function 217 the tissues. The binding of oxygen by hemoglobin is profoundly influenced by ph and CO 2 concentration, so the interconversion of CO 2 and bicarbonate is of great importance to the regulation of oxygen binding and release in the blood. Hemoglobin transports about 20% of the total H and CO 2 formed in the tissues to the lungs and the kidneys. The binding of H and CO 2 is inversely related to the binding of oxygen. At the relatively low ph and high CO 2 concentration of peripheral tissues, the affinity of hemoglobin for oxygen decreases as H and CO 2 are bound, and O 2 is released to the tissues. Conversely, in the capillaries of the lung, as CO 2 is excreted and the blood ph consequently rises, the affinity of hemoglobin for oxygen increases and the protein binds more O 2 for transport to the peripheral tissues. This effect of ph and CO 2 concentration on the binding and release of oxygen by hemoglobin is called the Bohr effect, after Christian Bohr, the Danish physiologist (and father of physicist Niels Bohr) who discovered it in The binding equilibrium for hemoglobin and one molecule of oxygen can be designated by the reaction v ph 7.4 ph 7.6 Hb O 2 34 HbO 2 ph 7.2 but this is not a complete statement. To account for the effect of H concentration on this binding equilibrium, we rewrite the reaction as HHb O 2 34 HbO 2 H where HHb denotes a protonated form of hemoglobin. This equation tells us that the O 2 -saturation curve of hemoglobin is influenced by the H concentration (Fig. 7 15). Both O 2 and H are bound by hemoglobin, but with inverse affinity. When the oxygen concentration is high, as in the lungs, hemoglobin binds O 2 and releases protons. When the oxygen concentration is low, as in the peripheral tissues, H is bound and O 2 is released. Oxygen and H are not bound at the same sites in hemoglobin. Oxygen binds to the iron atoms of the hemes, whereas H binds to any of several amino acid residues in the protein. A major contribution to the Bohr effect is made by His 146 (His HC3) of the b subunits. When protonated, this residue forms one of the ion pairs to Asp 94 (Asp FG1) that helps stabilize deoxyhemoglobin in the T state (Fig. 7 9). The ion pair stabilizes the protonated form of His HC3, giving this residue an abnormally high pk a in the T state. The pk a falls to its normal value of 6.0 in the R state because the ion pair cannot form, and this residue is largely unprotonated in oxyhemoglobin at ph 7.6, the blood ph in the lungs. As the concentration of H rises, protonation of His HC3 promotes release of oxygen by favoring a transition to the T state. Protonation of the amino-terminal residues of the a subunits, certain other His residues, and perhaps other groups has a similar effect. Thus we see that the four polypeptide chains of hemoglobin communicate with each other not only about O 2 binding to their heme groups, but also about H binding to specific amino acid residues. And there is still more to the story. Hemoglobin also binds CO 2, again in a manner inversely related to the binding of oxygen. Carbon dioxide binds as a carbamate group to the a-amino group at the amino-terminal end of each globin chain, forming carbaminohemoglobin: po 2 (kpa) figure 7 15 Effect of ph on the binding of oxygen to hemoglobin. The ph of blood is 7.6 in the lungs and 7.2 in the tissues. Experimental measurements on hemoglobin binding are often performed at ph 7.4. O B C B O N H 2 H A OCOCO A B R O Amino-terminal residue H O H H G A A CONOCO CO B A B O R O Carbamino-terminal residue

16 218 Part II Structure and Catalysis This reaction produces H, contributing to the Bohr effect. The bound carbamates also form additional salt bridges (not shown in Fig. 7 9) that help to stabilize the T state and promote the release of oxygen. When the concentration of carbon dioxide is high, as in peripheral tissues, some CO 2 binds to hemoglobin and the affinity for O 2 decreases, causing its release. Conversely, when hemoglobin reaches the lungs, the high oxygen concentration promotes binding of O 2 and release of CO 2. It is the capacity to communicate ligand-binding information from one polypeptide subunit to the others that makes the hemoglobin molecule so beautifully adapted to integrating the transport of O 2, CO 2, and H by erythrocytes. O G J C O A B HOCOOOPO A A HO COH O A O A OP A P O O O O 2,3-Bisphosphoglycerate Oxygen Binding to Hemoglobin Is Regulated by 2,3-Bisphosphoglycerate The interaction of 2,3-bisphosphoglycerate (BPG) with hemoglobin provides an example of heterotropic allosteric modulation. BPG is present in relatively high concentrations in erythrocytes. When hemoglobin is isolated, it contains substantial amounts of bound BPG, which can be difficult to remove completely. In fact, the O 2 -binding curves for hemoglobin that we have examined to this point were obtained in the presence of bound BPG. 2,3-Bisphosphoglycerate is known to greatly reduce the affinity of hemoglobin for oxygen there is an inverse relationship between the binding of O 2 and the binding of BPG. We can therefore describe another binding process for hemoglobin: HbBPG O 2 34 HbO 2 BPG BPG binds at a site distant from the oxygen-binding site and regulates the O 2 -binding affinity of hemoglobin in relation to the po 2 in the lungs. BPG plays an important role in the physiological adaptation to the lower po 2 available at high altitudes. For a healthy human strolling by the ocean, the binding of O 2 to hemoglobin is regulated such that the amount of O 2 delivered to the tissues is equivalent to nearly 40% of the maximum that could be carried by the blood (Fig. 7 16). If the same person is quickly transported to a mountainside at an altitude of 4,500 meters, where the po 2 is considerably lower, the delivery of O 2 to the tissues is reduced. However, po 2 in tissues po 2 in lungs (4500 m) po 2 in lungs (sea level) figure 7 16 Effect of BPG on the binding of oxygen to hemoglobin. The BPG concentration in normal human blood is about 5 mm at sea level and about 8 mm at high altitudes. Note that hemoglobin binds to oxygen quite tightly when BPG is entirely absent, and the binding curve appears to be hyperbolic. In reality, the measured Hill coefficient for O 2 -binding cooperativity decreases only slightly (from 3 to about 2.5) when BPG is removed from hemoglobin, but the rising part of the sigmoid curve is confined to a very small region close to the origin. At sea level, hemoglobin is nearly saturated with O 2 in the lungs, but only 60% saturated in the tissues, so that the amount of oxygen released in the tissues is close to 40% of the maximum that can be carried in the blood. At high altitudes, O 2 delivery declines by about one-fourth, to 30% of maximum. An increase in BPG concentration, however, decreases the affinity of hemoglobin for O 2 so that nearly 40% of what can be carried is again delivered to the tissues. v BPG = 0mM BPG 5 mm BPG 8 mm po 2 (kpa)

17 Chapter 7 Protein Function 219 (a) (b) after just a few hours at the higher altitude, the BPG concentration in the blood has begun to rise, leading to a decrease in the affinity of hemoglobin for oxygen. This adjustment in the BPG level has only a small effect on the binding of O 2 in the lungs but a considerable effect on the release of O 2 in the tissues. As a result, the delivery of oxygen to the tissues is restored to nearly 40% of that which can be transported by the blood. The situation is reversed when the person returns to sea level. The BPG concentration in erythrocytes also increases in people suffering from hypoxia, lowered oxygenation of peripheral tissues due to inadequate function of the lungs or circulatory system. BPG binds to hemoglobin in the cavity between the b subunits in the T state (Fig. 7 17). This cavity is lined with positively charged amino acid residues that interact with the negatively charged groups of BPG. Unlike O 2, only one molecule of BPG is bound to each hemoglobin tetramer. BPG lowers hemoglobin s affinity for oxygen by stabilizing the T state. The transition to the R state narrows the binding pocket for BPG, precluding BPG binding. In the absence of BPG, hemoglobin is converted to the R state more easily. Regulation of oxygen binding to hemoglobin by BPG has an important role in fetal development. Because a fetus must extract oxygen from its mother s blood, fetal hemoglobin must have greater affinity than the maternal hemoglobin for O 2. In fetuses, g subunits are synthesized rather than b subunits, and a 2 g 2 hemoglobin is formed. This tetramer has a much lower affinity for BPG than normal adult hemoglobin, and a correspondingly higher affinity for O 2. Sickle-Cell Anemia Is a Molecular Disease of Hemoglobin The great importance of the amino acid sequence in determining the secondary, tertiary, and quaternary structures of globular proteins, and thus their biological functions, is strikingly demonstrated by the hereditary human disease sickle-cell anemia. More than 300 genetic variants of hemoglobin are known to occur in the human population. Most of these variations consist of differences in a single amino acid residue. The effects on hemoglobin structure and function are often minor but can sometimes be extraordinary. Each hemoglobin variation is the product of an altered gene. The variant genes are called alleles. Because humans generally have two copies of each gene, an individual may have two copies of one allele (thus being homozygous for that gene) or one copy of each of two different alleles (c) figure 7 17 Binding of BPG to deoxyhemoglobin. (a) BPG binding stabilizes the T state of deoxyhemoglobin, shown here as a mesh surface image. (b) The negative charges of BPG interact with several positively charged groups (shown in blue in this GRASP surface image) that surround the pocket between the b subunits in the T state. (c) The binding pocket for BPG disappears on oxygenation, following transition to the R state. (Compare (b) and (c) with Fig )

18 220 Part II Structure and Catalysis (a) (heterozygous). Sickle-cell anemia is a genetic disease in which an individual has inherited the allele for sickle-cell hemoglobin from both parents. The erythrocytes of these individuals are fewer and also abnormal. In addition to an unusually large number of immature cells, the blood contains many long, thin, crescent-shaped erythrocytes that look like the blade of a sickle (Fig. 7 18). When hemoglobin from sickle cells (called hemoglobin S) is deoxygenated, it becomes insoluble and forms polymers that aggregate into tubular fibers (Fig. 7 19). Normal hemoglobin (hemoglobin A) remains solfigure 7 18 A comparison of uniform, cup-shaped, normal erythrocytes (a) with the variably shaped erythrocytes seen in sickle-cell anemia (b). These cells range from normal to spiny or sickle-shaped. 2 m (b) figure 7 19 Normal and sickle-cell hemoglobin. (a) Subtle differences between the conformations of hemoglobin A and hemoglobin S result from a single amino acid change in the b chains. (b) As a result of this change, deoxyhemoglobin S has a hydrophobic patch on its surface, which causes the molecules to aggregate into strands that align into insoluble fibers. Interaction between molecules Hemoglobin A Hemoglobin S b 1 a 2 Strand formation (a) b 2 a 1 Alignment and crystallization (fiber formation) (b)

19 Chapter 7 Protein Function 221 uble on deoxygenation. The insoluble fibers of deoxygenated hemoglobin S are responsible for the deformed sickle shape of the erythrocytes, and the proportion of sickled cells increases greatly as blood is deoxygenated. The altered properties of hemoglobin S result from a single amino acid substitution, a Val instead of a Glu residue at position 6 in the two b chains. The R group of valine has no electric charge, whereas glutamate has a negative charge at ph 7.4. Hemoglobin S therefore has two fewer negative charges than hemoglobin A, one for each of the two b chains. Replacement of the Glu residue by Val creates a sticky hydrophobic contact point at position 6 of the b chain, which is on the outer surface of the molecule. These sticky spots cause deoxyhemoglobin S molecules to associate abnormally with each other, forming the long, fibrous aggregates characteristic of this disorder. Sickle-cell anemia occurs in individuals homozygous for the sickle-cell allele of the gene encoding the b subunit of hemoglobin. Individuals who receive the sickle-cell allele from only one parent and are thus heterozygous experience a milder condition called sickle-cell trait; only about 1% of their erythrocytes become sickled on deoxygenation. These individuals may live completely normal lives if they avoid vigorous exercise or other stresses on the circulatory system. People with sickle-cell anemia suffer from repeated crises brought on by physical exertion. They become weak, dizzy, and short of breath, and they also experience heart murmurs and an increased pulse rate. The hemoglobin content of their blood is only about half the normal value of 15 to 16 g/100 m because sickled cells are very fragile and rupture easily; this results in anemia ( lack of blood ). An even more serious consequence is that capillaries become blocked by the long, abnormally shaped cells, causing severe pain and interfering with normal organ function a major factor in the early death of many people with the disease. Without medical treatment, people with sickle-cell anemia usually die in childhood. Nevertheless, the sickle-cell allele is surprisingly common in certain parts of Africa. Investigation into the persistence of an allele that is so obviously deleterious in homozygous individuals led to the finding that the allele confers a small but significant resistance to lethal forms of malaria in heterozygous individuals. Natural selection has resulted in an allele population that balances the deleterious effects of the homozygous condition against the resistance to malaria afforded by the heterozygous condition. Complementary Interactions between Proteins and igands: The Immune System and Immunoglobulins Our discussion of oxygen-binding proteins showed how the conformations of these proteins affect and are affected by the binding of small ligands (O 2 or CO) to the heme group. However, most protein-ligand interactions do not involve a prosthetic group. Instead, the binding site for a ligand is more often like the hemoglobin binding site for BPG a cleft in the protein lined with amino acid residues, arranged to render the binding interaction highly specific. Effective discrimination between ligands is the norm at binding sites, even when the ligands have only minor structural differences. All vertebrates have an immune system capable of distinguishing molecular self from nonself and then destroying those entities identified as nonself. In this way, the immune system eliminates viruses, bacteria, and other pathogens and molecules that may pose a threat to the organism. On a physiological level, the response of the immune system to an invader is an intricate and coordinated set of interactions among many classes of proteins,

20 222 Part II Structure and Catalysis molecules, and cell types. However, at the level of individual proteins, the immune response demonstrates how an acutely sensitive and specific biochemical system is built upon the reversible binding of ligands to proteins. table 7 2 Some Types of eukocytes Associated with the Immune System Cell type Function Macrophages Ingest large particles and cells by phagocytosis B lymphocytes Produce and secrete (B cells) antibodies T lymphocytes (T cells) Cytotoxic (killer) Interact with infected T cells (T C ) host cells through receptors on T-cell surface Helper T cells (T H ) Interact with macrophages and secrete cytokines (interleukins) that stimulate T C, T H, and B cells to proliferate. The Immune Response Features a Specialized Array of Cells and Proteins Immunity is brought about by a variety of leukocytes (white blood cells), including macrophages and lymphocytes, all arising from undifferentiated stem cells in the bone marrow. eukocytes can leave the bloodstream and patrol the tissues, each cell producing one or more proteins capable of recognizing and binding to molecules that might signal an infection. The immune response consists of two complementary systems, the humoral and cellular immune systems. The humoral immune system (atin humor, fluid ) is directed at bacterial infections and extracellular viruses (those found in the body fluids), but can also respond to individual proteins introduced into the organism. The cellular immune system destroys host cells infected by viruses and also destroys some parasites and foreign tissues. The proteins at the heart of the humoral immune response are soluble proteins called antibodies or immunoglobulins, often abbreviated Ig. Immunoglobulins bind bacteria, viruses, or large molecules identified as foreign and target them for destruction. Making up 20% of blood protein, the immunoglobulins are produced by B lymphocytes or B cells, so named because they complete their development in the bone marrow. The agents at the heart of the cellular immune response are a class of T lymphocytes or T cells (so called because the latter stages of their development occur in the thymus) known as cytotoxic T cells (T C cells, also called killer T cells). Recognition of infected cells or parasites involves proteins called T-cell receptors on the surface of T C cells. Recall from Chapter 2 (p. 30) that receptors are proteins, usually found on the outer surface of cells and extending through the plasma membrane; they recognize and bind extracellular ligands, triggering changes inside the cell. In addition to cytotoxic T cells, there are helper T cells (T H cells), whose function it is to produce soluble signaling proteins called cytokines, which include the interleukins. T H cells interact with macrophages. Table 7 2 summarizes the functions of the various leukocytes of the immune system. Each recognition protein of the immune system, either an antibody produced by a B cell or a receptor on the surface of a T cell, specifically binds some particular chemical structure, distinguishing it from virtually all others. Humans are capable of producing over 10 8 different antibodies with distinct binding specificities. This extraordinary diversity makes it likely that any chemical structure on the surface of a virus or invading cell will be recognized and bound by one or more antibodies. Antibody diversity is derived from random reassembly of a set of immunoglobulin gene segments via genetic recombination mechanisms that are discussed in Chapter 25. Some properties of the interactions between antibodies or T-cell receptors and the molecules they bind are unique to the immune system, and a specialized lexicon is used to describe them. Any molecule or pathogen capable of eliciting an immune response is called an antigen. An antigen may be a virus, a bacterial cell wall, or an individual protein or other macromolecule. A complex antigen may be bound by a number of different antibodies. An individual antibody or T-cell receptor binds only a particular molecular structure within the antigen, called its antigenic determinant or epitope. It would be unproductive for the immune system to respond to small molecules that are common intermediates and products of cellular metabolism. Molecules of M r 5,000 are generally not antigenic. However, small molecules can be covalently attached to large proteins in the laboratory, and in this

21 Chapter 7 Protein Function 223 form they may elicit an immune response. These small molecules are called haptens. The antibodies produced in response to protein-linked haptens will then bind to the same small molecules when they are free. Such antibodies are sometimes used in the development of analytical tests described later in this chapter or as catalytic antibodies (described in Box 8 3). The interactions of antibody and antigen are much better understood than are the binding properties of T-cell receptors. However, before focusing on antibodies, we need to look at the humoral and cellular immune systems in more detail to put the fundamental biochemical interactions into their proper context. Self Is Distinguished from Nonself by the Display of Peptides on Cell Surfaces The immune system must identify and destroy pathogens, but it must also recognize and not destroy the normal proteins and cells of the host organism the self. Detection of protein antigens in the host is mediated by MHC (major histocompatibility complex) proteins. MHC proteins bind peptide fragments of proteins digested in the cell and present them on the outside surface of the cell. These peptides normally come from the digestion of typical cellular proteins, but during a viral infection viral proteins are also digested and presented by MHC proteins. Peptide fragments from foreign proteins that are displayed by MHC proteins are the antigens the immune system recognizes as nonself. T-cell receptors bind these fragments and launch the subsequent steps of the immune response. There are two classes of MHC proteins (Fig. 7 20), which differ in their distribution among cell types and in the source of digested proteins whose peptides they display. figure 7 20 MHC proteins These proteins consist of a and b chains. In class I MHC proteins (a), the small b chain is invariant but the amino acid sequence of the a chain exhibits a high degree of variability, localized in specific domains of the protein that appear on the outside of the cell. Each human produces up to six different a chains for class 1 MHC proteins. In class II MHC proteins (b), both the a and b chains have regions of relatively high variability near their amino-terminal ends. Hypervariable domains a chain S S b chain H 3 N + S S a chain + NH 3 + NH 3 + NH 3 b chain S S S S S S S S OOC Extracellular space Plasma membrane OOC Cytosol OOC COO (a) Class I MHC protein (b) Class II MHC protein

22 224 Part II Structure and Catalysis figure 7 21 Structure of a human class I MHC protein. (a) This image is derived in part from the determined structure of the extracellular portion of the protein. The a chain of MHC is shown in gray; the small b chain is blue; the disulfide bonds are yellow. A bound ligand, a peptide derived from HIV, is shown in red. (b) Top view showing a surface contour image of the site where peptides are bound and displayed. The HIV peptide (red) occupies the site. This part of the class I MHC protein interacts with T-cell receptors. Class I MHC proteins (Fig. 7 21) are found on the surface of virtually all vertebrate cells. There are countless variants in the human population, placing them among the most polymorphic of proteins. Because individuals produce up to six class I MHC protein variants, any two individuals are unlikely to have the same set. Class I MHC proteins bind and display peptides derived from the proteolytic degradation and turnover of proteins that occurs randomly within the cell. These complexes of peptides and class I MHC proteins are the recognition targets of the T-cell receptors of the T C cells in the cellular immune system. The general pattern of immune system recognition was first described by Rolf Zinkernagel and Peter Doherty in Each T C cell has many copies of only one T-cell receptor that is specific for a particular class I MHC protein peptide complex. To avoid creating a Antigen a chain NH 3 NH 3 COO (b) b chain Extracellular space Plasma membrane Cytosol OOC (a)

23 Chapter 7 Protein Function 225 legion of T C cells that would set upon and destroy normal cells, the maturation of T C cells in the thymus includes a stringent selection process that eliminates more than 95% of the developing T C cells, including those that might recognize and bind class I MHC proteins displaying peptides from cellular proteins of the organism itself. The T C cells that survive and mature are those with T-cell receptors that do not bind to the organism s own proteins. The result is a population of cells that bind foreign peptides bound to class I MHC proteins of the host cell. These binding interactions lead to the destruction of parasites and virus-infected cells. When an organ is transplanted, its foreign class I MHC proteins are also bound by T C cells, leading to tissue rejection. Class II MHC proteins occur on the surfaces of a few types of specialized cells that take up foreign antigens, including macrophages and B lymphocytes. ike class I MHC proteins, the class II proteins are highly polymorphic, with many variants in the human population. Each human is capable of producing up to 12 variants, and thus it is unlikely that any two individuals have an identical set of variants. The class II MHC proteins bind and display peptides derived not from cellular proteins but from external proteins ingested by the cells. The resulting class II MHC protein peptide complexes are the binding targets of the T-cell receptors of the various helper T cells. T H cells, like T C cells, undergo a stringent selection process in the thymus, eliminating those that recognize the individual s own cellular proteins. Despite the elimination of most T C and T H cells during the selection process in the thymus, a very large number survive, and these provide the immune response. Each survivor has a single type of T-cell receptor that can bind to one particular chemical structure. The T cells patrolling the bloodstream and the tissues carry millions of different binding specificities in the T-cell receptors. Within the highly varied T-cell population there is almost always a contingent of cells that can specifically bind any antigen that might appear. The vast majority of these cells never encounter a foreign antigen to which they can bind and typically die within a few days, replaced by new generations of T cells endlessly patrolling in search of the interaction that will launch the full immune response. Molecular Interactions at Cell Surfaces Trigger the Immune Response A new antigen is often the harbinger of an infection a signal to the immune system that a virus or other parasite may be rapidly growing in the organism. Those few T cells and B cells possessing receptors or antibodies that can bind the antigen must be rapidly and selectively propagated to eliminate the infection. A hypothetical viral infection illustrates how this occurs. When a virus invades a cell, it makes use of cellular functions and resources to replicate its nucleic acid and make viral proteins. Once inside the cell, viral macromolecules are relatively inaccessible to the antibodies of the humoral immune system. However, some of the class I MHC proteins that find their way to the surface of an infected cell will generally display peptide fragments from viral proteins, which can then be recognized by T C lymphocytes. Mature viruses become vulnerable to the humoral immune system when they are released from the infected cell and are present for a time in the extracellular environment. Some are then ingested by macrophages (which ingest only those antigens that are recognized by the antibodies produced by a particular B cell). Viral peptide fragments will be displayed on the surfaces of the macrophages and B cells, complexed to class II MHC proteins, and the peptide antigens will trigger a multi-pronged response involving B cells, T C cells, and T H cells (Fig. 7 22).

24 MHC class I protein displaying cellular peptides. Host cell Viral pathogens MHC I T-cell receptor MHC II CD4 CD8 Interleukin receptor Interleukin 2, 4 ysed cell releasing virus particles and antigens; some become bound by antibodies. Infected cell with MHC I displaying normal host and viral peptides. Soluble antibodies Infected cell with T C cell bound to MHC I and viral peptides via T-cell receptor and CD8 receptor protein. T-cell receptor CD8 Interleukin receptor T C cell IgG Fc receptor Antibody-bound virus being engulfed by macrophage. Viral peptides presented by MHC II. CD4 B cell B cell that produces antibodies to virus. T-cell receptor T H cell T H cell Interleukin 2 B cell Interleukin 4 Infected cell destroyed by T C cell. T C cell T C and T H cells bound to infected cells mature and proliferate, stimulated by interleukins. Memory cells T C, T H, B Macrophage with MHC II complexed to T H cell via T-cell receptor and CD4. B cell with MHC II and viral peptides complexed with T H cells via T-cell receptor and CD4. B cell proliferates and produces soluble antibodies, stimulated by interleukins. figure 7 22 Overview of the immune response to a viral infection. The individual steps are described in the text. 226

25 Chapter 7 Protein Function 227 The class I MHC protein peptide complexes on infected cells are recognized as foreign and bound by those T C cells with T-cell receptors having the appropriate binding specificity. The T-cell receptors respond only to peptide antigens that are complexed to class I MHC proteins. The T C cells have an additional receptor, CD8, also called a coreceptor, that enhances the binding interactions of T-cell receptors and MHC proteins (Fig. 7 22, middle left). The T C cells live up to the name killer T cells by destroying the virally infected cell to which they are complexed through their T-cell receptors. Cell death is brought about by a number of mechanisms, not all well understood. One mechanism involves the release of a protein called perforin, which binds to and aggregates in the plasma membrane of the target cell, forming molecular pores that destroy the capacity of that cell to regulate its interior environment. T C cells also induce a process called programmed cell death, or apoptosis (most commonly pronounced app -a-toe -sis), in which the cells complexed to T C cells undergo metabolic changes that rapidly lead to the demise of the cell. T C cells with the proper specificity must proliferate selectively if large numbers of virus-infected cells are to be destroyed. To this end, T C cells complexed to an infected cell generate cell-surface receptors for signaling proteins called interleukins. Interleukins, secreted by a variety of cells, stimulate the proliferation of only those T and B cells bearing the required interleukin receptors. Because T and B cells produce interleukin receptors only when they are complexed with an antigen, the only immune system cells that proliferate are those few that can respond to the antigen. The process of producing a population of cells by stimulated reproduction of a particular ancestor cell is called clonal selection. The peptides complexed to class II MHC proteins and displayed on the surface of macrophages and B lymphocytes are similarly bound by the appropriate T-cell receptors of T H cells. The T H cells also have a coreceptor, called CD4, that enhances the binding interactions of the T-cell receptors. This overall binding interaction, in concert with secondary molecular signals that are currently being identified, activates the T H cells. A subpopulation of activated T H cells secrete a small signal protein called interleukin-2 (I-2; M r 15,000), which stimulates proliferation of nearby T C cells and T H cells having the appropriate interleukin receptors. This greatly increases the number of available immune system cells capable of recognizing and responding to the antigen. Another subpopulation of activated T H cells complexed to macrophages or B lymphocytes secrete interleukin-4 (I-4; M r 20,000), which stimulates the proliferation of B cells that recognize the antigen (Fig. 7 22, bottom right). Proliferation of the responding B, T C, and T H cells continues as long as the appropriate antigen is present. The proliferating B cells promote the destruction of any extracellular viruses or bacterial cells. They first secrete large amounts of soluble antibody that binds to the antigen. This bound antibody recruits a cellular system of about 20 proteins collectively called complement because they complement and enhance the action of the antibodies. The complement proteins disrupt the coats of many viruses or, in bacterial infections, produce holes in the cell walls of bacteria, causing them to swell and burst by osmotic shock. Unlike T cells, B cells do not undergo selection in the thymus to eliminate those producing antibodies that recognize host (self) proteins. However, B cells do not contribute significantly to an immune response unless they are stimulated to proliferate by T H cells. The T H cells do undergo selection in the thymus, leaving no T H cells capable of stimulating B cells that produce antibodies potentially dangerous to the host. The T H cells themselves participate only indirectly in the destruction of infected cells and pathogens, but their role is critical to the entire immune

26 228 Part II Structure and Catalysis figure 7 23 The structure of immunoglobulin G. (a) Pairs of heavy and light chains combine to form a Y-shaped molecule. Two antigen-binding sites are formed by the combination of variable domains from one light (V ) and one heavy (V H ) chain. Cleavage with papain separates the Fab and Fc portions of the protein in the hinge region. The Fc portion of the molecule also contains bound carbohydrate. (b) A ribbon model of the first complete IgG molecule to be crystallized and structurally analyzed. Although the molecule contains two identical heavy chains (two shades of blue) and two identical light chains (two shades of red), it crystallized in the asymmetric conformation shown. Conformational flexibility may be important to the function of immunoglobulins. Antigenbinding site H 3 N + + NH 3 V H Papain cleavage sites V H H 3 N Antigenbinding site NH response. This is dramatically illustrated by the epidemic produced by HIV (human immunodeficiency virus), the virus that causes AIDS (acquired immune deficiency syndrome). The primary targets of HIV infection are T H cells. Elimination of these cells progressively incapacitates the entire immune system. Once antigen is depleted, activated immune cells generally die in a matter of days by programmed cell death. However, a few of the stimulated B and T cells mature into memory cells. These are long-lived cells that do not participate directly in the primary immune response when the antigen is first encountered. Instead they become permanent residents of the blood, ready to respond to a reappearance of the same antigen. Memory cells, when subsequently challenged by the antigen, can mount a secondary immune response that is generally much more rapid and vigorous than the primary response because of prior clonal expansion. By this mechanism, vertebrates once exposed to a virus or other pathogen can respond quickly to the pathogen when exposed again. This is the basis of the long-term immunity conferred by vaccines and the natural immunity to repeated infections by the same strain of a virus. Antibodies Have Two Identical Antigen-Binding Sites Immunoglobulin G (IgG) is the major class of antibody molecule and one of the most abundant proteins in the blood serum. IgG has four polypeptide chains: two large ones, called heavy chains, and two light chains, linked by noncovalent and disulfide bonds into a complex of M r 150,000. The heavy chains of an IgG molecule interact at one end, then branch to interact separately with the light chains, forming a Y-shaped molecule (Fig. 7 23). At the hinges separating the base of an IgG molecule from its branches, the immunoglobulin can be cleaved with proteases. Cleavage with the protease papain liberates the basal fragment, called Fc because it usually crystallizes readily, and the two branches, which are called Fab, the antigen-binding fragments. Each branch has a single antigen-binding site. Fab V C H 1 C H 1 V C OOC S S S S S S S S COO C Fc C H 2 C H 3 Bound carbohydrate C H 3 C H 3 OOC COO C = constant domain V = variable domain H, = heavy, light chains (a) (b)

27 Chapter 7 Protein Function 229 The fundamental structure of immunoglobulins was first established by Gerald Edelman and Rodney Porter. Each chain is made up of identifiable domains; some are constant in sequence and structure from one IgG to the next, others are variable. The constant domains have a characteristic structure known as the immunoglobulin fold, a well-conserved structural motif in the all-b class. There are three of these constant domains in each heavy chain and one in each light chain. The heavy and light chains also have one variable domain each, in which most of the variability in amino acid residue sequence is found. The variable domains associate to create the antigen-binding site (Fig. 7 24). figure 7 24 Binding of IgG to an antigen. To generate an optimal fit for the antigen, the binding sites of IgG often undergo slight conformational changes. Such induced fit is common to many protein-ligand interactions. H 3 N + + NH 3 H 3 N NH OOC S S S S S S S S COO Antigen S S S S S S S S OOC COO Antibody Antigen-antibody complex In many vertebrates, IgG is only one of five classes of immunoglobulins. Each class has a characteristic type of heavy chain, denoted a, d, e, g, and m for IgA, IgD, IgE, IgG, and IgM, respectively. Two types of light chain, k and l, occur in all classes of immunoglobulins. The overall structures of IgD and IgE are similar to that of IgG. IgM occurs in either a monomeric, membrane-bound form or a secreted form that is a cross-linked pentamer of this basic structure (Fig. 7 25). IgA, found principally in secretions such as saliva, tears, and milk, can be a monomer, dimer, or trimer. IgM is the first antibody to be made by B lymphocytes and is the major antibody in the early stages of a primary immune response. Some B cells soon begin to produce IgD (with the same antigen-binding site as the IgM produced by the same cell), but the unique function of IgD is less clear. m Heavy chains J chain ight chains figure 7 25 IgM pentamer of immunoglobulin units. The pentamer is cross-linked with disulfide bonds. The J chain is a polypeptide of M r 20,000 found in both IgA and IgM.

28 230 Part II Structure and Catalysis The IgG described above is the major antibody in secondary immune responses, which are initiated by memory B cells. As part of the organism s ongoing immunity to antigens already encountered and dealt with, IgG is the most abundant immunoglobulin in the blood. When IgG binds to an invading bacterium or virus, it not only activates the complement system, but also activates certain leukocytes such as macrophages to engulf and destroy the invader. Yet another class of receptors on the cell surface of macrophages recognizes and binds the Fc region of IgG. When these Fc receptors bind an antibody-pathogen complex, the macrophage engulfs the complex by phagocytosis (Fig. 7 26). figure 7 26 Phagocytosis of an antibody-bound virus by a macrophage. The Fc regions of the antibodies bind to Fc receptors on the surface of the macrophage, triggering the macrophage to engulf and destroy the virus. Phagocytosis IgG-coated virus Fc region of IgG IgG Fc receptor Macrophage IgE plays an important role in the allergic response, interacting with basophils (phagocytic leukocytes) in the blood and histamine-secreting cells called mast cells that are widely distributed in tissues. This immunoglobulin binds, through its Fc region, to special Fc receptors on the basophils or mast cells. In this form, IgE serves as a kind of receptor for antigen. If antigen is bound, the cells are induced to secrete histamine and other biologically active amines that cause dilation and increased permeability of blood vessels. These effects on the blood vessels are thought to facilitate the movement of immune system cells and proteins to sites of inflammation. They also produce the symptoms normally associated with allergies. Pollen or other allergens are recognized as foreign, triggering an immune response normally reserved for pathogens. Antibodies Bind Tightly and Specifically to Antigen The binding specificity of an antibody is determined by the amino acid residues in the variable domains of its heavy and light chains. Many residues in these domains are variable, but not equally so. Some, particularly those lining the antigen-binding site, are hypervariable especially likely to differ. Specificity is conferred by chemical complementarity between the antigen and its specific binding site, in terms of shape and the location of charged, nonpolar, and hydrogen-bonding groups. For example, a binding site with a negatively charged group may bind an antigen with a positive charge in the complementary position. In many instances, complementarity is achieved interactively as the structures of antigen and binding site are influenced by each other during the approach of the ligand. Conformational changes in the antibody and/or the antigen then occur that allow the complementary groups to interact fully. This is an example of induced fit (Fig. 7 27).

29 Chapter 7 Protein Function 231 Conformation with no antigen bound (a) Antigen bound (hidden) (b) Antigen bound (shown) (c) A typical antibody-antigen interaction is quite strong, characterized by K d values as low as M (recall that a lower K d corresponds to a stronger binding interaction). The K d reflects the energy derived from the various ionic, hydrogen-bonding, hydrophobic, and van der Waals interactions that stabilize the binding. The binding energy required to produce a K d of M is about 65 kj/mol. A complex of a peptide derived from HIV (a model antigen) and an Fab molecule illustrates some of these properties (Fig. 7 27). The changes in structure observed on antigen binding are particularly striking in this example. figure 7 27 Induced fit in the binding of an antigen to IgG. The molecule, shown in surface contour, is the Fab fragment of an IgG. The antigen this IgG binds is a small peptide derived from HIV. Two residues from the heavy chain (blue) and one from the light chain (pink) are colored to provide visual points of reference. (a) View of the Fab fragment looking down on the antigen-binding site. (b) The same view, but here the Fab fragment is in the bound conformation; the antigen has been omitted from the image to provide an unobstructed view of the altered binding site. Note how the binding cavity has enlarged and several groups have shifted position. (c) The same view as in (b), but with the antigen pictured in the binding site as a red stick structure. The Antibody-Antigen Interaction Is the Basis for a Variety of Important Analytical Procedures The extraordinary binding affinity and specificity of antibodies makes them valuable analytical reagents. Two types of antibody preparations are in use: polyclonal and monoclonal. Polyclonal antibodies are those produced by many different B lymphocytes responding to one antigen, such as a protein injected into an animal. Cells in the population of B lymphocytes produce antibodies that bind specific, different epitopes within the antigen. Thus, polyclonal preparations contain a mixture of antibodies that recognize different parts of the protein. Monoclonal antibodies, in contrast, are synthesized by a population of identical B cells (a clone) grown in cell culture. These antibodies are homogeneous, all recognizing the same epitope. The techniques for producing monoclonal antibodies were developed by Georges Köhler and Cesar Milstein. The specificity of antibodies has practical uses. A selected antibody can be covalently attached to a resin and used in a chromatography column of the type shown in Figure 5 18c. When a mixture of proteins is added to the column, the antibody will specifically bind its target protein and retain it on the column while other proteins are washed through. The target protein can then be eluted from the resin by a salt solution or some other agent. This is a powerful tool for protein purification. In another versatile analytical technique, an antibody is attached to a radioactive label or some other reagent that makes it easy to detect. When the antibody binds the target protein, the label reveals the presence of the protein in a solution or its location in a gel or even a living cell. Several variations of this procedure are illustrated in Figure Georges Köhler Cesar Milstein

30 232 Part II Structure and Catalysis figure 7 28 Antibody techniques. The specific reaction of an antibody with its antigen is the basis of several techniques that identify and quantify a specific protein in a complex sample. (a) A schematic representation of the general method. (b) An EISA testing for the presence of herpes simplex virus (HSV) antibodies in blood samples. Wells were coated with an HSV antigen, to which antibodies against HSV in a patient s blood will bind. The second antibody is anti human IgG linked to horseradish peroxidase. Blood samples with greater amounts of HSV antibody turn brighter yellow. (c) An immunoblot. anes 1 to 3 are from an SDS gel; samples from successive stages in the purification of a protein kinase have been separated and stained with Coomassie blue. anes 4 to 6 show the same samples, but these were electrophoretically transferred to a nitrocellulose membrane after separation on an SDS gel. The membrane was then probed with antibody against the protein kinase. The numbers between the gel and the immunoblot indicate M r ( 10 3 ). An EISA (enzyme-linked immunosorbent assay) allows for rapid screening and quantification of the presence of an antigen in a sample (Fig. 7 28b). Proteins in a sample are adsorbed to an inert surface, usually a 96-well polystyrene plate. The surface is washed with a solution of an inexpensive nonspecific protein (often casein from nonfat dry milk powder) to block proteins in subsequent steps from also adsorbing to these surfaces. The surface is then treated with a solution containing the primary antibody an antibody against the protein of interest. Unbound antibody is washed away and the surface is treated with a solution containing antibodies against the primary antibody. These secondary antibodies have been linked to an enzyme that catalyzes a reaction that forms a colored product. After unbound secondary antibody is washed away, the substrate of the antibodylinked enzyme is added. Product formation (monitored as color intensity) is proportional to the concentration of the protein of interest in the sample. In an immunoblot assay (Fig. 7 28c), proteins that have been separated by gel electrophoresis are transferred electrophoretically to a nitrocellulose membrane. The membrane is blocked (as described above for EISA), then treated successively with primary antibody, secondary antibody linked to enzyme, and substrate. A colored precipitate forms only along the band containing the protein of interest. The immunoblot allows 1 Coat surface with sample (antigens). 2 Block unoccupied sites with nonspecific protein. 3 Incubate with primary antibody against specific antigen. 4 Incubate with antibody-enzyme complex that binds primary antibody. 5 Add substrate. 6 Formation of colored product indicates presence of specific antigen. (a) EISA assay SDS gel Immunoblot (b) (c)

31 Chapter 7 Protein Function 233 the detection of a minor component in a sample and provides an approximation of its molecular weight. We will encounter other aspects of antibodies in later chapters. They are extremely important in medicine and can tell us much about the structure of proteins and the action of genes. Protein Interactions Modulated by Chemical Energy: Actin, Myosin, and Molecular Motors Organisms move. Cells move. Organelles and macromolecules within cells move. Most of these movements arise from the activity of the fascinating class of protein-based molecular motors. Fueled by chemical energy, usually derived from ATP, large aggregates of motor proteins undergo cyclic conformational changes that accumulate into a unified, directional force the tiny force that pulls apart chromosomes in a dividing cell, and the immense force that levers a pouncing, quarter-ton jungle cat into the air. The interactions among motor proteins, as you might predict, feature complementary arrangements of ionic, hydrogen-bonding, hydrophobic, and van der Waals interactions at protein binding sites. In motor proteins, however, these interactions achieve exceptionally high levels of spatial and temporal organization. Motor proteins underlie the contraction of muscles, the migration of organelles along microtubules, the rotation of bacterial flagella, and the movement of some proteins along DNA. As we noted in Chapter 2, proteins called kinesins and dyneins move along microtubules in cells, pulling along organelles or reorganizing chromosomes during cell division (see Fig. 2 19). An interaction of dynein with microtubules brings about the motion of eukaryotic flagella and cilia. Flagellar motion in bacteria involves a complex rotational motor at the base of the flagellum (see Fig ). Helicases, polymerases, and other proteins move along DNA as they carry out their functions in DNA metabolism (Chapter 25). Here, we focus on the wellstudied example of the contractile proteins of vertebrate skeletal muscle as a paradigm for how proteins translate chemical energy into motion. 20 nm 2 nm Carboxyl terminus (a) ight meromyosin Two supercoiled α helices trypsin 150 nm Tail Myosin Amino terminus Heavy meromyosin + papain ight chains 17 nm Heads The Major Proteins of Muscle Are Myosin and Actin The contractile force of muscle is generated by the interaction of two proteins, myosin and actin. These proteins are arranged in filaments that undergo transient interactions and slide past each other to bring about contraction. Together, actin and myosin make up over 80% of the protein mass of muscle. Myosin (M r 540,000) has six subunits: two heavy chains (M r 220,000) and four light chains (M r 20,000). The heavy chains account for much of the overall structure. At their carboxyl termini, they are arranged as extended a helices, wrapped around each other in a fibrous, left-handed coiled coil similar to that of a-keratin (Fig. 7 29a). At its amino termini, each heavy chain has a large globular domain containing a site where ATP is hydrolyzed. The light chains are associated with the globular domains. (b) S2 S1 S1 figure 7 29 Myosin. (a) Myosin has two heavy chains (in two shades of pink), the carboxyl termini forming an extended coiled coil (tail) and the amino termini having globular domains (heads). Two light chains (blue) are associated with each myosin head. (b) Cleavage with trypsin and papain separates the myosin heads (S1 fragments) from the tails. (c) Ribbon representation of the myosin S1 fragment. The heavy chain is in gray, the two light chains in two shades of blue. (c)

32 234 Part II Structure and Catalysis figure 7 30 The major components of muscle. (a) Myosin aggregates to form a bipolar structure called a thick filament. (b) F-actin is a filamentous assemblage of G-actin monomers that polymerize two by two, giving the appearance of two filaments spiraling about one another in a right-handed fashion. An electron micrograph and a model of F-actin are shown. (c) Space-filling model of an actin filament (red) with one myosin head (gray and two shades of blue) bound to an actin monomer within the filament. When myosin is treated briefly with the protease trypsin, much of the fibrous tail is cleaved off, dividing the protein into components called light and heavy meromyosin (Fig. 7 29b). The globular domain, called myosin subfragment 1, or S1, or simply the myosin head group, is liberated from heavy meromyosin by cleavage with papain. The S1 fragment produced by this procedure is the motor domain that makes muscle contraction possible. S1 fragments can be crystallized, and their structure has been determined. The overall structure of the S1 fragment as determined by Ivan Rayment and Hazel Holden is shown in Figure 7 29c. In muscle cells, molecules of myosin aggregate to form structures called thick filaments (Fig. 7 30a). These rodlike structures serve as the core of the contractile unit. Within a thick filament, several hundred myosin molecules are arranged with their fibrous tails associated to form a long bipolar structure. The globular domains project from either end of this structure, in regular stacked arrays. The second major muscle protein, actin, is abundant in almost all eukaryotic cells. In muscle, molecules of monomeric actin, called G-actin (globular actin; M r 42,000), associate to form a long polymer called F-actin ( filamentous actin). The thin filament (Fig. 7 30b) consists of F-actin, along with the proteins troponin and tropomyosin. The filamentous parts of thin filaments assemble as successive monomeric actin molecules add to one end. On addition, each monomer binds ATP, then hydrolyzes it to ADP, so all actin molecules in the filament are complexed to ADP. However, this ATP hydrolysis by actin functions only in the assembly of the filaments; it does not contribute directly to the energy expended in muscle contraction. Each actin monomer in the thin filament can bind tightly and specifically to one myosin head group (Fig. 7 30c). ~325 nm (a) Myosin head G-actin subunits 36 nm (b) Actin filament (c)

33 Chapter 7 Protein Function 235 Additional Proteins Organize the Thin and Thick Filaments into Ordered Structures Skeletal muscle consists of parallel bundles of muscle fibers, each fiber a single, very large, multinucleated cell, 20 to 100 mm in diameter, formed from many cells fused together and often spanning the length of the muscle. Each fiber, in turn, contains about 1,000 myofibrils, 2 mm in diameter, each consisting of a vast number of regularly arrayed thick and thin filaments complexed to other proteins (Fig. 7 31). A system of flat membranous vesicles called the sarcoplasmic reticulum surrounds each myofibril. Examined under the electron microscope, muscle fibers reveal alternating regions of high and low electron density, called the A and I bands (Fig. 7 31b,c). The A and I bands arise from the arrangement of figure 7 31 Structure of skeletal muscle. (a) Muscle fibers consist of single, elongated, multinucleated cells that arise from the fusion of many precursor cells. Within the fibers are many myofibrils (only six are shown here for simplicity) surrounded by the membranous sarcoplasmic reticulum. The organization of thick and thin filaments in the myofibril gives it a striated appearance. When muscle contracts, the I bands narrow and the Z disks come closer together, as seen in electron micrographs of relaxed (b) and contracted (c) muscle. (a) Bundle of muscle fibers Myofibrils Nuclei Capillaries Muscle Fiber Muscle Myofibril Sarcoplasmic reticulum I band Sarcomere A band Z disk M line (b) I band A band (c) Z disk M line Z disk